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This invention relates to the field of xenotransplanation and immunotolerance. More specifically, it relates to conjugates of galactose xcex11,3 galactosyl epitope(s) (xcex1Gal) for use in reducing levels of circulating anti-xcex1Gal antibodies and inducing immune tolerance to xenotransplanted tissue.
There is a large and increasing need for organ transplantation, which is exacerbated by a critical shortage of available human organs for transplant. The possibility of employing xenotransplantation to overcome the lack of human organs for allotransplantation is a possible solution to the critical organ shortage but in itself presents serious problems.
Two basic types of xenografts have been studied. In concordant xenografts, an organ from a donor animal is transplanted into a similar species which lacks antibodies to the donor organ. Rejection of a concordant organ is usually caused by T cell-mediated reactivity to differences in major histocompatability antigens. Concordant xenotransplantation had been applied to human patients as early as 1963 with the most celebrated case being the transplantation of a baboon heart into a human neonate. Auchincloss et al. (1998) Ann. Rev. Immunol. 16:433. In discordant transplants, the donor and recipient are phylogenetically more distant and the recipient has antibodies to the donor organ as is the case of porcine organs transplanted into Old World primates. These xenografts are rejected within the first few minutes due to the phenomenon of hyperacute rejection (HAR).
In humans and Old World primates, natural antibodies specific for the galactose xcex11,3 galactosyl (xcex1Gal) epitope mediate both the hyperacute rejection (HAR) and delayed xenograft rejection (DXR) of organs xenotransplanted from animals such as pigs. Humans and Old World primates express high levels of circulating antibodies to galactose xcex11,3 galactosyl (xcex1Gal) residues which are expressed at high levels on membrane lipids and proteins of other animal species. Galili et al. (1988) J. Biol. Chem. 263:17755. This is due to the fact that humans and Old World primates lack the enzyme xcex11,3 galactosyltransferase which is required to express the xcex1Gal epitope (Sandrin et al. (1993) Proc. Natl. Acad. Sci. USA 90:11391) and thus make antibodies to the xcex1Gal epitope which is expressed on normal gut flora and is recognized as foreign. By contrast, xenograft organ donor species (e.g., pigs) express the xcex11,3 galactosyltransferase enzyme and thus express the xcex1Gal epitope. It has been estimated that there are approximately 107 xcex1Gal epitopes per cell on many donor tissue cells. Cooper et al. (1998) Xenotransplantation 5:6-17.
When an xcex1Gal-expressing discordant organ is transplanted into a recipient that produces anti-xcex1Gal antibodies, the first deleterious result is HAR mediated by the high levels (up to 4% of circulating IgM) of the natural anti-xcex1Gal Ig. Parker (1994) J. Immunol. 153:3791. xcex1Gal bearing transplants (porcine islets) into humans have been shown to increase anti-xcex1Gal responses by up to 64-fold. Galili et al. (1995) Transplantation 59:1549. Transplantation of pig cartilage into cynomolgus monkeys causes a 30-300 fold increase in IgG anti-xcex1Gal and 2-16 fold increase in IgM. Galili et al. (1997) Transplantation 63:646. These natural antibodies bind to xcex1Gal expressed on the endothelial surfaces of the engrafted organ which both activates the complement system and the endothelium causing damage to the cell and sets up a net prothrombotic state on the vessel surface. The result is a damaged endothelium and massive clotting causing the rapid development of ischemia of the transplanted organ and its functional incapacitation within minutes to hours.
Anti-xcex1Gal antibodies also play a role in delayed xenograft rejection (DXR), the intermediate term (several days) damage to xenotransplanted organs which often results in rejection. Bach et al. (1995) Nature Med. 1:869-873. Anti-xcex1Gal antibodies bind to the endothelium and mediate a plethora of cell-mediated responses such as ADCC, leading to vascular and tissue damage which compromises the function of the transplanted organ. Anti-xcex1Gal antibodies can also cause chronic activation of receptors on xenograft cells and/or increased immunogenicity of xenograft-specific antigens. Cooper et al. (1998) Xenotransplantation 5:6-17. Diminution of anti-xcex1Gal antibodies levels can attenuate this damage, but the damage, even if not acute, is cumulative and can eventually leads to organ rejection.
Various strategies have been pursued to deal with the major sequelae of anti-xcex1Gal antibody binding. These include: 1) inhibition of effector functions mediated, directly or indirectly by anti-xcex1Gal antibodies; 2) depletion of recipient anti-xcex1Gal antibodies; 3) sublethal irradiation and reconstitution of recipient with autologous and donor bone marrow; 4) modification of donor tissue glycosylation; 5) suppression of anti-xcex1Gal antibodies in recipient; and 6) administration of xcex1Gal moieties found on donor tissue to the recipient.
In attempts to inhibit the effector functions mediated by anti-xcex1Gal antibody binding, much effort has been spent to generate donor animals transgenic for complement regulatory proteins such as decay accelerating factor (DAF, CD55), and homologous restriction factor (HRF, CD59). Schmoekel et al. (1996) Transplantation 62:729; and Byrne et al. (1997) Transplantation 63:149. These proteins, members of the regulators of complement activation (RCA) gene family, can downregulate the generation of complement proteins which mediate acute inflammation and cell lytic activity. Transgenic expression of RCA proteins does nothing, however, to address the effect of anti-xcex1Gal antibodies binding to the endothelium which sets up the net prothrombotic state subsequent to endothelial cell activation. Thus, while RCA-transgenic donor organs may have an ameliorative effect on HAR, this approach will not have an effect on DXR wherein xenograft damage is mediated by effector cells which bind to anti-xcex1Gal antibodies. Further, while the expression of RCA molecules on the transplanted organ may protect the transplant from protective functions of the host/recipient immune response, if the transplanted organ were to express pathogen antigens (from viral or bacterial infection), protective antibody effector function mediated by complement would be effectively shut off leaving the host with an organ full of pathogen-infected cells. For these reasons, strategies aimed at controlling complement are thought to be insufficient to achieve long-term graft survival unless acceptable regimens are developed to reduce antibody responses. Soulillou (1998) Xenotransplantation 5:1-2.
Depleting the recipient of pathogenic anti-xcex1Gal antibodies has been shown to prolong pig organ survival. Cooper et al. (1996) Xenotransplantation 4:27. However, attempts to remove the natural anti-xcex1Gal antibodies by apheresis have been only temporarily effective and result in an only slightly delayed antibody-mediated organ rejection. Pathogenic levels of anti-xcex1Gal antibodies return within 1-2 days and mediate xenograft damage. Sablinski et al. (1995) Xenotransplantation 2:264; Cooper et al. (1998) Xenotransplantation 5:6. Thus, very frequent ex vivo plasmapheresis to reduce the incidence of HAR would be required. Furthermore, the cumulative damage by levels of anti-xcex1Gal antibodies too low to mediate HAR but which mediate DXR are unavoidable using the plasmapheresis strategy. In addition, currently available pharmacological interventions, even in combination, only have modest effect in inhibiting anti-xcex1Gal antibody production. Cooper et al. (1998) Xenotransplantation 5:6. Thus, it seems unlikely that permanent suppression of anti-xcex1Gal responses will be possible using this approach.
The use of mixed chimerism, wherein human organ recipients would be sublethally irradiated and reconstituted with autologous and porcine bone marrow, has been contemplated. xcex1Gal transferase knock out mice which do not express xcex1Gal have been tolerized via mixed hematopoietic chimerism. Thall et al. (1998) J. Exp. Med. However, instituting microchimerism of porcine marrow cells in primates would be problematic since primate marrow does not have the requisite species-specific hematopoietic cytokines/growth factors to facilitate their engraftment in the primate marrow.
Attempts to modify glycosylation of donor tissue, such as pig vascular endothelium have also been contemplated. However, pig knockouts (KO) created to effect the glycosylation modifications have not yet been accomplished due to a lack of porcine embryonic stem cells. Homologous recombination leading to the elimination of the xcex11,3 galactosyl transferase enzyme which leads to the expression of the xcex1Gal moiety has been accomplished in mice. Thall et al. (1995) J. Biol. Chem. 270:21437. xcex1Gal knock-out hearts still undergo rapid rejection when perfused with human plasma in ex vivo systems indicating that decreasing xcex1Gal production may increase other xenoantigens such as the Forssman antigen. Tange et al. (1997) Xenotransplantation 4:20. In addition, extreme modifications of carbohydrate antigen expression on cell surfaces may have a detrimental effect on development and differentiation. Attempts to decrease xcex1gal expression by making animals transgenic for a fucosyl transferase does not sufficiently decrease antigen expression. Galili et al. (1998) Science and Medicine 9:28. Thus, the general approach of modifying xcex1Gal expression, while theoretically appealing, may be untenable.
PCT application WO 98US2103 generally describes xcex1Gal compositions for inducing tolerance. French patent application FR 2751346 (based on WO 9803653) describes transgenic cells for transplantation which contain polynucleotides which encode antibodies directed against molecules involved in rejection. PCT application WO 9716064 describes transgenic cells which express a functional carbohydrate epitope modifying gene product which modifies a cell surface carbohydrate epitope such as xcex1Gal. See also PCT WO 950661 (nucleic acid sequences encoding xcex11,3-galactosyltransferase) and PCT WO 9421799 (nucleic acid sequences encoding xcex11,3-galactosyltransferase).
In sum, in humans and Old World primates, natural antibodies specific for the galactose xcex11,3 galactosyl (xcex1Gal) epitope mediate both the hyperacute rejection (HAR) and delayed xenograft rejection (DXR) of organs xenotransplanted from animals such as pigs. Anti-xcex1Gal-mediated complement activation and endothelial cell activation induce an acute net procoagulant state on the endothelium of the engrafted organ leading to rapid clotting, ischemia and loss of organ function. Attempts to remove the natural anti-xcex1Gal antibodies have been only temporarily effective and result in an only slightly delayed antibody-mediated organ rejection.
There is a serious need to develop compositions (such as toleragens) and methods that counteract the effect of circulating xcex1Gal antibodies in order to promote effectiveness of xenotransplantation.
All publications cited herein are hereby incorporated by reference in their entirety.
We have discovered xcex1Gal conjugates with significantly increased ability to bind to anti-xcex1Gal antibodies, including IgM antibodies, which are the major effector molecules of hyperacute rejection. The invention thus provides these conjugates, compositions comprising these conjugates, and methods using these conjugates.
Accordingly, in one aspect, the invention provides a conjugate comprising a valency platform molecule and an xcex1Gal epitope, wherein the valency platform molecule has a valency between two and 128. Preferred embodiments include valency platform molecules with a valency of two, four, six, eight, ten, twelve, sixteen, twenty, and twenty-five. The xcex1Gal epitope may be any moiety which specifically binds to an anti-xcex1Gal antibody.
In another aspect, the invention provides a conjugate comprising a valency platform molecule and an xcex1Gal epitope, wherein the valency platform molecule has a valency of at least two, four, six, eight, ten, 12, 16, 20, and 25. The xcex1Gal epitope may be any moiety which specifically binds to an anti-xcex1Gal antibody.
In another aspect, the invention provides compositions comprising the conjugates described herein, and preferably contain an effective amount of any of the conjugates described herein. In some embodiments, the compositions comprise a pharmaceutical excipient.
In another aspect, the invention provides methods of reducing circulating levels of anti-xcex1Gal antibodies in an individual, comprising administering an effective amount any of the conjugates described herein to the individual, wherein an effective amount is an amount sufficient to reduce the circulating levels of anti-xcex1Gal antibodies.
In another aspect, the invention provides methods of neutralizing circulating levels of anti-xcex1Gal antibodies in an individual, comprising administering an effective amount of any of the conjugates described herein to the individual, wherein an effective amount is an amount sufficient to neutralize circulating levels of anti-xcex1Gal antibodies.
In another aspect, the invention provides methods of inducing immunological tolerance (generally to a xenotransplantation antigen, more specifically to xcex1Gal), comprising administering an effective amount any of the conjugates described herein to the individual.
In another aspect, the invention provides methods of detecting the presence and/or amount of anti-xcex1Gal antibody in a biological sample comprising (a) contacting an xcex1Gal conjugate of the invention with the biological sample under conditions that permit the formation of a stable antigen-antibody complex; and (b) detecting stable complex, if any, formed in step (a).
In another aspect, the invention provides methods for performing a xenotransplantation in an individual, comprising the steps of: (a) administering any of the conjugate(s) or composition(s) described herein to the individual; and (b) introducing xenotissue to an individual.
In another aspect, the invention provides methods of suppressing rejection of a transplanted tissue comprising an xcex1 Gal epitope in an individual, said method comprising administering any of the conjugates described herein to the individual in an amount sufficient to suppress rejection.